Test system for the determination of the presence of an antibiotic in a fluid

ABSTRACT

Provided is a test system, a test method and a test kit based on a test medium comprising Bromothymol Blue or a structurally related indicator.

FIELD OF THE INVENTION

The present invention relates to an improved novel microbiological testsystem and a new method for the rapid determination of the presence ofantibacterial compounds in fluids such as milk, meat juice, serum andurine using said test system.

BACKGROUND OF THE INVENTION

Microbiological test methods for the determination of antibacterialcompounds, particularly residues of antibiotics such as cephalosporin,penicillin, tetracycline and derivatives thereof and chemotherapeuticssuch as sulfa's, in fluids such as milk, meat juice, serum and urinehave been known for a long time. Examples of such tests have beendescribed in CA 2056581, DE 3613794, EP 0005891, EP 0285792, EP 0611001,GB A 1467439 and U.S. Pat. No. 4,946,777. These descriptions all dealwith ready to use tests that make use of a microorganism and will give aresult by the change indicated by an indicator molecule added to thetest system. The principle is that when an antibacterial compound ispresent in the fluid in a concentration sufficient to inhibit the growthof the microorganism the color of the indicator will stay the same,while, when no inhibition occurs, the growth of the microorganism isaccompanied by the formation of acid or reduced metabolites or otherphenomena that will induce an indicator signal.

The test systems mentioned above include a test medium, such as an agarmedium, inoculated with a microorganism, preferably a strain ofBacillus, Escherichia coli or Streptococcus, and a pH indicator and/or aredox indicator. The microorganism and the indicator are introduced intoan optionally buffered agar solution, optionally nutrients are added tothe solution and optionally substances to change the sensitivity tocertain antimicrobial compounds are added to the solution. Finally theagar solution is allowed to solidify to form the test medium such thatthe microorganisms stay alive but cannot multiply because of lack ofnutrients and/or low temperature. A suitable test should have thedesired sensitivity with regard to the compounds to be tested for.

The problem with the test systems currently distributed on the marketand/or described in literature is that they have a limited sensitivitytowards certain antibiotics. One of the consequences of this problem isthat for certain applications, for instance when threshold requirementsare changed, an adequate test system cannot be made available with thecurrent technology. There is thus a need for an improved test methodthat does not have this problem.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an improved methodfor the determination of antibiotics in fluids. Surprisingly, we havefound that there is a positive effect attainable when applying theindicator according to the invention.

By applying the indicator of the present invention in microbiologicaltest systems, an advantage in sensitivity towards antibiotics, such asfor instance β-lactams and aminoglycosides can be achieved. By applyingsaid indicator in a method for the determination of antibiotics influids, increases in sensitivity can be achieved. Said increases canamount up to 25% and even up to 100% depending on the antibiotic inquestion. Additionally, it has been found that the use of said indicatoralso results in a test system showing an improved visual contrast whencomparing positive and negative samples. This latter phenomenon greatlyfacilitates accurate visual evaluation of test results.

The present invention provides a test system for the determination ofthe presence of an antibiotic in a fluid which comprises a test mediumcomprising a microorganism, a substance that provides a solid state andan indicator suitable for the detection of penicillin G, characterizedin that said indicator is a compound having the general formula:

wherein R₁, R₂, R₃ and R₄ are independently of each other alkyl, halogenor hydrogen, X═C or S, n=1 if X═C and n=0, 1 or 2 if X═S, and R₅ and R₆are independently of each other:

wherein R₇, R₈ and R₉ are, independently of each other alkyl, branchedalkyl, hydrogen or halogen and R₁₀ is alkyl or branched alkyl, or saltsthereof.

Furthermore, there is provided a method for the determination of thepresence of an antibiotic in a fluid comprising the steps of:

-   -   (a) contacting a sample of said fluid with a test medium        comprising a micro-organism, at least one substance that        provides a solid state and an indicator;    -   (b) incubating the microorganism for a period of time to grow        the microorganism in case no antibiotic is present in the fluid        sample; and    -   (c) detecting growth or inhibition of growth of the        microorganism with the indicator, characterized in that said        indicator is a compound having the general formula (I).

Furthermore, there is provided a kit suitable for the determination ofan antibiotic in a fluid comprising a container partially filled with atest medium comprising a micro-organism, a gelling agent and anindicator, characterized in that said indicator is a compound with thegeneral formula (I).

Finally there is provided the use of a compound having the generalformula (I) as indicator in a test system for an antibiotic.

DETAILED DESCRIPTION OF THE INVENTION

The terms and abbreviations given below are used throughout thisdisclosure and are defined as follows.

The term ‘CFU’ is an abbreviation of Colony Forming Units and refers tothe number of microorganisms, spores of microorganisms, partiallygerminated spores of microorganisms or vegetative cells capable ofproducing colonies of microorganisms.

The term ‘fluid’ refers to a substance (as a liquid) tending to flow orconform to the outline of its container.

The term ‘gelling agent’ refers to a compound that assists in changing amixture into or taking on the form of a gel.

The term ‘indicator’ refers to a substance used to measure (for exampleby change of color or fluorescence) the condition of a test medium withrespect to the presence of a particular material (for example an acid, abase, oxidizing or reducing agents). For instance, the term ‘indicator’may refer to one or more compounds that are known as pH-indicators, butalso to one or more compounds that are known as redox-indicators. Also,the term ‘indicator’ may refer to mixtures of two or more differenttypes of indicators, such as a combination of a pH- and aredox-indicator. In general, when two or more indicators are used, theseindicators are co-operating to increase the indicator effect of each ofthe indicators when taken alone.

The term ‘nutrient’ refers to one or more nutritive substances oringredients that promote and/or are required for the growth ofmicroorganisms as used in the method of the present invention.

The term ‘sampling device’ refers to a device with the aid of which asample of a fluid can be added to a test medium. Such a device may be acontainer, optionally with volume markings. Such a container may be acapillary, a syringe, a pipette or an automated pipetting system. Such asyringe or pipette may be designed in such a way that with only one modeof operation a predetermined volume can be withdrawn from the fluid tobe analyzed.

The term ‘sensitivity’ refers to the degree of receptiveness of a givensystem to sense a certain state. More, particularly, in the present case‘sensitivity’ refers to the degree by which concentrations ofantibiotics in a sample can be determined.

The term ‘spore’ refers to a primitive usually unicellular oftenenvironmentally resistant dormant or reproductive body produced bymicroorganisms and capable of development into a new individualmicroorganism.

The term ‘test medium’ refers to a composition such as a solution, asolid or, preferably, in the form of a sol or a gel, for instancecomprising a gelling agent. Suitable examples of gelling agents areagar, alginic acid and salts thereof, carrageenan, gelatin,hydroxypropylguar and derivatives thereof, locust bean gum (Carob gum),processed eucheuma seaweed and the like. However, the person skilled inthe art will understand that other types of solid test media may bebased on carrier materials such as ceramics, cotton, glass, metalparticles, paper, polymers in any shape or form, silicates, sponges,wool and the like. Usually, a test medium contains one or moreindicators, however, these compounds may also be added later when thetest is being performed. The test medium comprises one or more types ofmicroorganisms as detecting agents. Optionally, the test medium may alsocontain one or more buffers, nutrients, stabilizers, substances thatchange the sensitivity to certain antimicrobial compounds in a positiveor negative way, and/or viscosity-increasing agents. When a buffer ispresent in the medium, it may be added during the mixing of thecomponents of the medium or the components may be dissolved and/orsuspended in the buffer. Optionally the test medium is sterilized andusually the pH is adjusted to the required value. Examples of substancesthat change the sensitivity to certain antimicrobial compounds areantifolates like ormethoprim, tetroxoprim and trimethoprim that improvethe sensitivity of the micro-organism towards sulfa compounds or saltsof oxalic acid or hydrofluoric acid, which improve the sensitivitytowards tetracycline. Examples of viscosity-increasing agents areascorbyl methylsilanol pectinate, carbomer, carboxymethyl cellulose,cetearyl alcohol, cetyl alcohol, cetyl esters, cocamide DEA, emulsifyingwax, glucose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose,lauramide DEA, linoleamide DEA, magnesium aluminum silicate,maltodextrins, PEG-8 distearate, polyacrylamide, polyvinyl alcohol,PVP/hexadecene copolymer, sodium chloride, sodium sulfate,soyamidopropyl betaine, xanthan gum and the like. Alternatively, theoptional ingredients of the test medium mentioned above may also beadded exogenously. The test medium may be contained within any type ofcontainer; frequently used containers are tubes, microtiter plates andpetri dishes.

The term ‘threshold’ refers to the concentration value above which agiven analyte is to be regarded as present and below which said analyteis to be regarded as absent. Generally, a threshold value is given forparticular analytes in particular samples by local, regional orinterregional authorities but it can also be pre-set for certainresearch purposes.

In a first aspect of the invention there is provided a test system thatcomprises a test medium. The test medium comprises a microorganism, asubstance that provides a solid state and at least one indicator, atleast one of which has the general formula (I) given above. Preferredexamples of said indicator are Bromocresol Green, Bromothymol Blue,Chlorocresol Green, m-Cresol Purple, Thymol Blue and Xylenol Blue. Mostpreferably the indicator is Bromothymol Blue.

Preferably, the substance providing for a solid state is a gelling agentand/or a carrier material. The amount of gelling agent in the testmedium is between 2 and 100 g.l⁻¹, preferably between 5 and 50 g.l⁻¹,more preferably between 10 and 20 g.l⁻¹, most preferably between 12 and15 g.l⁻¹. Preferably the gelling agent is agar.

In an embodiment of the first aspect of the invention, the microorganismis a thermo stable microorganism such as a Bacillus species, preferablyBacillus stearothermophilus, an Escherichia coli species, or aStreptococcus species, preferably Streptococcus thermophilus. Thesespecies may be introduced in the test as units capable of producingcolonies, or Colony Forming Units (CFU's). Said CFU's may be spores,vegetative cells or a mixture of both. The concentration of said CFU'sis expressed as Colony Forming Units per ml of test medium (CFU.ml⁻¹)and is usually in the range of 1×10⁵ to 1×10¹² CFU.ml⁻¹, preferably1×10⁶ to 1×10¹⁰ CFU.ml⁻¹, more preferably 2×10⁶ to 1×10⁹ CFU.ml⁻¹, mostpreferably 5×10⁶ to 1×10⁸ CFU.ml⁻¹, or still more preferably 5×10⁶ to2×10⁷ CFU.ml⁻¹.

In a second aspect of the invention, there is provided a method for thedetermination of an antibiotic in a fluid comprising the steps ofcontacting a sample of said fluid with a test medium according to thefirst aspect of the present invention in the presence of nutrients.Advantageously, the method provides for conditions that there is nogrowth of microorganism prior to the addition of fluid sample, bykeeping the test medium at conditions that prevent growth, such as arelatively low temperature and/or in the absence of nutrients essentialfor growth. After addition of the fluid sample, growth of themicroorganism is allowed to take place during a period sufficiently longfor the microorganisms to grow in case no antibiotics are present, byadding nutrients, optionally before the contacting of said fluid sample,and/or raising the temperature, and/or providing for a pH-value at whichthe microorganism is able to grow; and detecting growth of themicroorganism by observing the presence or absence of a change of anindicator. The method of the present invention also includes mixingsamples (e.g. with other samples, but also with salts, bufferingcompounds, nutrients, stabilizers, isotope-labeled compounds,fluorescence-labeled compounds and the like), concentrating and/ordiluting (e.g. with diluting liquids such as water, milk or liquidsderived from milk, blood or liquids derived from blood, urine and/orsolvents) samples prior to addition to the test medium.

In one embodiment of the second aspect of the present invention, theantibiotic is a β-lactam antibiotic such as a cephalosporin or apenicillin derivative. Examples of such derivatives are amoxicillin,ampicillin, cefadroxil, cefradine, ceftiofur, cephalexin, penicillin G,penicillin V and ticarcillin, but of course many other similar β-lactamderivatives are known and applicable in the method of the presentinvention. In another embodiment the antibiotic is an aminoglycosidesuch as, for instance, neomycin.

Advantageously, it was established that the method of the presentinvention displays selectivity with regard to antibiotics, in particularwith regard to β-lactam antibiotics and aminoglycosides.

In another embodiment of the second aspect of the invention, the growthof the microorganism is to take place during a predetermined period,preferably within a time span of 0.5 to 4 hours, more preferably between1 to 3.5 hours, most preferably between 2.0 to 3.25 hours. Preferablythe growth of the microorganism is conducted at a predeterminedtemperature, preferably the optimal growth temperature of themicro-organism. When, for example, thermo stable microorganisms areused, said temperature preferably is between 40 and 70° C., morepreferably between 50 and 65° C., most preferably between 60 and 64° C.Optionally said reaction can be carried out with the aid of athermostatic device. Alternatively, the time required for growth of themicro-organism is equal to the time that is required for a calibrationsample without any analyte to induce a change in the indicator.

In still another embodiment of the second aspect of the invention,nutrients are added as a separate source, e.g. as a tablet, disc or apaper filter. Also other compounds such as the indicator(s),microorganism, stabilizers and/or antifolates may be added as a separatesource, optionally incorporated in the nutrient medium.

In yet another embodiment of the second aspect of the invention, thereis provided a method for determining the presence or absence of anantibiotic in a fluid sample whereby the ratio of the fluid sample totest medium exceeds 2:3 (0.68:1) (v/v). Preferably, said ratio is atleast 20:27 (0.74:1) (v/v), more preferably said ratio is at least 25:27(0.93:1) (v/v); most preferably said ratio is at least 2:1 (v/v). It hasbeen found that there is no technical reason for an upper limit to theamount of fluid sample. In practice this volume should not exceed themaximum content of the container that holds the test medium. Forexample, in a 2 ml container having 0.2 ml test medium, no more than 1.8ml of fluid sample should be added. In practice, containers forperforming the method of the present invention have a volume that rarelyexceeds 50 ml and hence the amount of fluid sample to be added shall notexceed 50 ml, preferably 10 ml, more preferably 5 ml, still morepreferably 2 ml, most preferably 1 ml. Thus, in general, the upper limitof the ratio of the volume of fluid sample to the volume of test mediumis 250:1 (v/v), preferably 50:1 (v/v), more preferably 25:1 (v/v), stillmore preferably 10:1 (v/v), most preferably 5:1 (v/v). Preferably, thevolume of fluid sample is greater than the volume of test medium.

The result of the method of the present invention is determined by theobservation of the presence or absence of a change of the indicator orindicators used. When, for example such a change is a color change, saidcolor change may be observed visually. However in one embodiment of theinvention said color change is determined using an arrangement thatgenerates digital image data or an arrangement that generates analogimage data and converts said analog image data into digital image datafollowed by interpretation of said digital image data by a computerprocessor. Such an arrangement, which may for instance be asample-reading device such as a scanner coupled to a personal computer,is described in International Patent Application WO 03/033728,incorporated by reference, and briefly summarized below.

The arrangement can be suitably used for instance for detecting residuesof antibiotics in milk. With this arrangement it is possible to scan thebottom side of each of the samples in a test plate. The color and thebrightness of the reflected light are registered in three variables,each describing one color component, for instance the so-called L*a*b*model. In the L*a*b* model, the color spectrum is divided in atwo-dimensional matrix. The position of a color in this matrix isregistered by means of the two variables “a” and “b”. The variable Lindicates the intensity (for instance, from light blue to dark-blue). Itis possible to make a criterion comprising the a-value, b-value andL-value to make a composite function as follows:Z=w _(L) ·L+w _(a) ·a+w _(b) ·b

where w_(L), w_(a) and w_(b) are weighting factors for the L-value,a-value and b-value, respectively. The values of these weighting factorscan be calculated by means of “discriminent analysis”, such that thegroup means show a maximum distance in relation to the spreading. Bycombining two or more of the color components in the L*a*b* model in apredetermined manner that depends on the type of residue and the sample,an accurate detection is possible. In practice, a certain value of Z atwhich a test should switch between positive and negative result isexperimentally predetermined.

In a third aspect of the invention there is provided a kit for carryingout the method of the second aspect of the present invention. Such a kitcomprises one or more containers filled with test medium as described inthe first aspect of the invention and optionally a sampling device. Thecontainers may be test tubes of any shape and size and from any materialavailable, provided that observation of indicator changes is possible.Also, the containers may be wells such as those incorporated inmicro-titer plates.

Said sampling device is a device with the aid of which fluid can beadded to said test medium. Preferably, such a device is a container,optionally with volume markings. More preferably, such a device is asyringe, a pipette or an automated pipetting system. Such a syringe orpipette may be designed in such a fashion that with only one mode ofoperation a predetermined volume can be withdrawn from the fluid to beanalyzed. Optionally, systems known in the art with which more than onesyringe or pipette can be operated with one single handling may beapplied. It is the object of the second aspect of the present inventionto provide a kit that allows for simple addition of the amounts of fluidto be added according the first aspect of the invention. Optionally,said kit comprises means for sealing of said containers filled with testmedium during incubation and/or an insert with instructions for useand/or a means for setting the time needed for incubation.

In one embodiment of the third aspect of the invention, said kitcomprises nutrients. Preferably said nutrients are contained within amedium such as a tablet, disc or a paper filter. The advantages ofproviding nutrients contained within a medium are that the user caneasily add them to the test medium and that the amounts can bepredetermined so as to avoid errors in dosing the required amounts. Alsoother compounds such as the indicator(s), stabilizers and/or antifolatesmay be added as a separate source, optionally incorporated in thenutrient medium.

In another embodiment of the third aspect of the present invention, saidkit comprises a thermostatic device, With the aid of which test samplescan be kept at a pre-set temperature, such as the temperature at whichthe microorganism shows sufficient growth. Preferably, said thermostaticdevice is designed in such a fashion that it can hold said containersfilled with test medium. Optionally the thermostatic device is coupledto a means for setting the time needed for incubation such that heatingand/or cooling is stopped after lapse of a pre-set period.

In yet another embodiment of the third aspect of the invention, said kitcomprises a data carrier loaded with a computer program suitable forinstructing a computer to analyze digital data obtained from asample-reading device. Said data carrier may be any carrier suitable forstoring digital information such as a CD-ROM, a diskette, a DVD, amemory stick, a magnetic tape or the like. Advantageously, said datacarrier loaded with a computer program provides for easy access to thelatest available computer programs suitable for use in the method of thepresent invention.

In a fourth aspect of the present invention there is provided the use ofa compound having the general formula (I) to improve the sensitivity foran antibiotic in a microbial inhibition test.

EXAMPLES Example 1 Comparison of Bromothymol Blue and Bromocresol Purplein Penicillin G Determination

Commercially, available Delvotest® MCS, prepared according to themethods described in EP 0005891, was adapted by the replacement of theindicator Bromocresol Purple by Bromothymol Blue (160 mg.l⁻¹). Thesensitivity for penicillin G was determined by investigating samplescontaining different concentrations of penicillin G in different testsystems. The results are summarized in the Table below (sensitivityvalues in ppb). Bromocresol Purple Bromothymol Blue Penicillin G¹ 4 2Penicillin G² 3 2¹Penicillin G concentrations of 0, 2, 4 and 6 ppb were investigated²Penicillin G concentrations of 0, 1, 2, 3 and 4 ppb were investigated

Example 2

Comparison of Bromothymol Blue and Bromocresol Purple in Determinationof Various Antibiotics

Commercially, available Delvotest® MCS, prepared according to themethods described in EP 0005891, was adapted by the replacement of theindicator Bromocresol Purple by Bromothymol Blue ('160 mg.l⁻¹). Thesensitivity for different antibiotics was determined by investigatingtwo sets of six experiments using either a plate test or a tube testsystem. The sensitivity was determined by reading the test at the momentat which an antibiotic-free control changed color. From the results assummarized in the Table below (sensitivity values in ppb), it can beseen that Bromothymol Blue gives superior sensitivities in comparisonwith Bromocresol purple (BP) for all antibiotics investigated with theexception of sulfadiazine in which case the results for the twoindicators are identical. Bromothymol Blue Type of test BP¹ Plate testTube test Experiment # 0 1 2 3 4 5 6 7 8 9 10 11 12 Penicillin G 3 1 2 11 1-2 1 2 2 2 2 2 2 Ampicillin 8 2 2 2 2 2 2 2 2 2 2 2 2 Amoxicillin 8 42 2 2 2-4 2 4 4 4 4 4 4 Cephapirin 8 2 2 4 6 2 4-6 4 4 4 4 4 4Cloxacillin 40 10 10 10 10 10 10 20 20 20 20 20 20 Neomycin 600 100 100100 100 100 100 200 200 200 200 200 200 Sulfadiazine 50 50 50 50 50 5050 50 50 50 50 50 50¹BP: Bromocresol Purple

Example 3 Concentration Effect in the Comparison of Bromothymol Blue andBromocresol Purple

Two test systems were prepared. The first system differs only from acommercially available Delvotest® MCS in that the concentration ofBromocresol Purple is 160 mg.l⁻¹, and the second series is as the firstwith Bromocresol Purple replaced with Bromothymol Blue (160 mg.l⁻¹). Thesensitivity for penicillin G was determined reading the test at themoment at which an antibiotic-free control changed color. From theresults as shown in the Table below (sensitivity values in ppb) it canbe seen that the sensitivity of Bromothymol Blue is better than that ofBromocresol Purple at the same concentrations of indicator. BromocresolPurple Bromothymol Blue (160 mg · l⁻¹) (160 mg · l⁻¹) Penicillin G 3 2

Example 4 Comparison of Bromothymol Blue and Bromocresol Purple inProduction Scale Microbiological Inhibition Test

Two series of test systems were prepared on production scale. The firstseries is a commercially available Delvotest® MCS and the second seriesis as the first with Bromocresol Purple replaced with Bromothymol Blue(160 mg.l⁻¹). The sensitivity for different antibiotics was determinedreading the test at the moment at which an antibiotic-free controlchanged color. The results are in the Table below (sensitivity values inppb). Bromocresol Purple Bromothymol Blue Penicillin G¹ 3 2 PenicillinG² 3 2 Ampicillin² 6 2 Amoxicillin² 8 4 Cephapirin² 8 4 Cloxacillin² 4020 Neomycin² 400 200¹Batch #1, immediately after production²Batch #1, three weeks after production

1. Test system for the determination of the presence of an antibiotic ina fluid comprising a test medium comprising a microorganism, at leastone substance that provides a solid state and an indicator suitable forthe detection of penicillin G, characterized in that said indicator is acompound having the general formula:

wherein R₁, R₂, R₃ and R₄ are independently of each other alkyl, halogenor hydrogen, X=C or S, n=1 if X═C and n=0, 1 or 2 if X═S, and R₅ and R₆are independently of each other:

wherein R₇, R₈ and R₉ are, independently of each other alkyl, branchedalkyl, hydrogen or halogen and R₁₀ is alkyl or branched alkyl, or saltsthereof.
 2. Test system according to claim 1 wherein R₁₀ is methyl. 3.Test system according to claim 2 wherein said indicator is BromothymolBlue.
 4. Method for the determination of the presence of an antibioticin a fluid comprising the steps of: a) contacting a sample of said fluidwith a test medium comprising a micro-organism, at least one substancethat provides a solid state and an indicator; (b) incubating themicroorganism for a period of time to grow the microorganism in case noantibiotic is present in the fluid sample; and (C) detecting growth orinhibition of growth of the microorganism with the indicator,characterized in that said test system is a test system according toclaim
 1. 5. Method according to claim 4 wherein the antibiotic to bedetermined is a β-lactam antibiotic.
 6. Method according to claim 4wherein the fluid in which antibiotics are to be determined is a fluidobtainable from an animal or human body.
 7. Method according to claim 4wherein the ratio of the volume of said fluid to the volume of testmedium exceeds 0.68:1.
 8. A method according to claim 4, wherein theratio of the volume of liquid sample to the volume of test mediumexceeds 20:27 (0.74:1) (v/v), 25:27 (0.93:1) (v/v) or 2:1 (v/v).
 9. Amethod according to claim 4, wherein the volume of liquid sample isgreater than the volume of test medium.
 10. Kit suitable for thedetermination of an antibiotic in a fluid comprising a containerpartially filled with a test medium comprising a microorganism, agelling agent and an indicator, characterized in that said indicator isa compound with the general formula (I).
 11. Kit according to claim 8further comprising nutrients suitable for allowing the microorganism togrow.
 12. Kit according to claim 10, further comprising a thermostaticdevice, with the aid of which test samples can be kept at a pre-settemperature.
 13. Kit according to claim 10, further comprising a datacarrier loaded with a computer program suitable for instructing acomputer to analyze digital data obtained from a sample-reading device.14. Use of a compound having the general formula (I) as indicator in atest system for an antibiotic.
 15. Use according to claim 14 wherein theantibiotic is a β-lactam antibiotic.